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DOI: 10.1055/s-0033-1352344
Molecular cloning and functional characterization of an NADPH:cytochrome P450 reductase from a tropical medicinal plant Scoparia dulcis
Cytochrome P450 monooxygenases (P450 s) are commonly involved in biosynthesis of endogenous compounds and catabolism of xenobiotics. All known plant P450 reactions depend on the associated activity of an NADPH:cytochrome P450 reductase (CPR) that catalyzes the transfer of electrons from NADPH via FAD and FMN to the prosthetic heme group of the P450 protein. We isolated a cDNA for CPR gene (designated as SdCPR) from Scoparia dulcis leaf. The SdCPR cDNA contains an open reading frame encoding a protein of 713 amino acids, with a predicted relative molecular weight of 78.5 kD. By aligning deduced sequence of SdCPR with other plant CPRs from taxonomically diverse species all functional domains involved in the binding of the P450, and cofactors of FMN, FAD and NADPH, were identified. Amino acid sequence comparison showed that the Scoparia CPR share high sequence identities with CPRs from other flowering plant species (66 – 80%). It belong to the Class I of dicotyledonous CPR. To characterize the activity of SdCPR, enzyme was produced in Escherichia coli as recombinant protein. After solubilization from membrane and affinity purification, the purity of the SdCPR protein was verified via SDS-polyacrylamide gel electrophoresis. Strong cytochrome c reductase activated were observed for SdCPR when NADPH was added. By contrast, NADH did not support either SdCPR for the reducing reactions, indicating that SdCPR utilize NADPH specifically as the electron donor. Compared to the electron donor (NADPH), the requirement of CPR for electron acceptor is relatively less specific: cytochrome c, K3Fe(CN)6, and dichlorophenolindophenol (DCPIP) can all serve as acceptor. Currently, we are evaluating the ability of SdCPR to support P450 activity using an Arabidopsis P450, CYP73A5. The expression of SdCPR in Scoparia plant was investigated by real-time PCR, which showed that transcripts of SdCPR were present in all tissues examined, including leaf, stem and root.